HUMAN MONOCLONAL-ANTIBODY HA-1A BINDS TO ENDOTOXIN VIA AN EPITOPE IN THE LIPID-A DOMAIN OF LIPOPOLYSACCHARIDE

HA-1A, a human IgM mAb, has been shown to significantly reduce mortali ty in septic patients with Gram-negative bacteremia, especially those with septic shock, in a controlled clinical trial. To confirm the repo rted specificity of this antibody for the lipid A domain of endotoxin, several assay systems were developed. These assay systems included an ELISA, which measured the binding of HA-1A to lipid A adsorbed to a s olid phase; a rate nephelometry assay, which measured the ability of H A-1A to bind and aggregate lipid A in solution; and a dot-blot immunoa ssay, which measured the ability of HA-1A to interact with lipid A ads orbed to Immobilon-P. In all three assay systems, HA-1A bound in a dos e-dependent. manner to lipid A prepared from Salmonella minnesota R595 LPS, whereas negative control human IgM mAb or polyclonal antibodies did not. Several experimental approaches were employed to demonstrate the specificity of HA-1A in these assay systems. Both polymyxin B and murine IgG mAb (8A1) with a specificity for lipid A were able to compe titively inhibit HA-1A reactivity with lipid A in a dose-dependent man ner. Furthermore, a murine IgG anti-Id mAb (9B5.5) developed against H A-1A was also able to block the binding of HA-1A to lipid A in these a ssay formats. HA-1A reactivity with synthetic lipid A confirmed that H A-1A binding to the natural lipid A was not the result of contaminants in the latter. Finally, the reactivity of HA-1A against a variety of glucosamine-containing and fatty acid-containing compounds was assesse d. Some weak interaction was seen with cardiolipin and chitin, but not with serum proteins, lipoteichoic acid, or DNA. Collectively, these r esults conclusively establish that HA-1A binds to the lipid A region o f LPS by an interaction with the V region of the antibody.